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Results for “HIV Long Terminal Repeat”

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PubMed1995

Cross-linking of Fc gamma receptors activates HIV-1 long terminal repeat-driven transcription in human monocytes.

Elevation of the levels of circulating immune complexes frequently accompanies HIV-1 infection and is a prognostic indicator of clinical progression from asymptomatic infection to AIDS. Here we report that cross-linking of Fc gamma RI or Fc gamma RII by adherent human IgG or by specific anti-Fc gamma R mAb activates HIV-1 gene expression in the human monocytic cell line BF24 and increased HIV RNA expression in monocytes from HIV infected patients as assayed by reverse transcription-PCR. In THP-1 cells, Fc gamma R cross-linking induced NF-kappa B, which is known to bind to the regulatory region of the long terminal repeat (LTR) of HIV-1 and to activate HIV-1 transcription. Anti-TNF-alpha antibody but not anti-IL-1 beta antibody strongly inhibited both the induction of HIV-1-LTR-driven transcription and the induction of NF-kappa B by Fc gamma R cross-linking. These results indicate that Fc gamma R can mediate a TNF-alpha-dependent induction of HIV-1 gene transcription and suggest that immune complexes may contribute to the pathophysiology of HIV-1 infection by augmenting viral replication in monocytes.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Secretion of extracellular factor(s) induced by X-irradiation activates the HIV type 1 long terminal repeat through its kappaB motif.

X-irradiation has been used in the treatment of several human diseases, including AIDS-related-malignancies. X-irradiation might induce the transcription and the replication of human immunodeficiency virus type 1 (HIV-1) and enhance nuclear factor kappa B (NF-kappaB). In the present article we show that the activation of the HIV-1 long terminal repeat (LTR) by direct X-irradiation can be mimicked by coculture of transfected cells with X-irradiated nontransfected (HIV-1-negative) cells. In the human colonic carcinoma cell line HT29, the activation seems to depend on an extracellular factor(s) released by a cell line treated with X-rays. The HIV-1 LTR cis-acting element conferring X-indirect responsiveness was identified as the kappaB tandem motif. The two main nuclear HIV-1 kappaB-binding complexes activated by X-direct and -indirect irradiation were the NF-kappaB p50/p65 and c-Rel/p65 heterodimers. Nuclear NF-kappaB activation was dependent on protein neosynthesis. It was partially inhibited by 100 microM pyrrolidine dithiocarbamate, a potent antioxidant drug, but was not correlated with a significant decrease in cellular IkappaBalpha. Furthermore, X-irradiation induces the expression of several cytokine genes generally associated with stress response and antibodies against interleukin 6 and TNF-alpha partially inhibited the X-indirect activation of the HIV-1 LTR. The use of protein kinase C (PKC)-specific inhibitor and of forskolin, an adenylate cyclase activator, suggests that a PKC-dependent pathway and the cAMP intracellular concentration could play a role in the X-indirect enhancement of HIV-1 LTR transcription in the HT29 cell line. In addition, supernatants of an X-irradiated HT29 cell culture activated the HIV-1 stimulation in infected peripheral blood monocytes.

Antioxidants↗

PubMedDate not supplied

Human herpesvirus 8 viral FLICE-inhibitory protein inhibits Fas-mediated apoptosis through binding and prevention of procaspase-8 maturation.

Viral FLICE-inhibitory proteins (v-FLIPs) encoded by several herpesviruses and poxviruses share the ability to inhibit apoptosis after engagement of death receptors. In the current article, we provide insights into the mechanisms by which the v-FLIP of human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated virus) protects cells from apoptosis after Fas-induced signaling. Using v-FLIP expression vectors, our results clearly show that HHV-8 v-FLIP reduces the cleavage of procaspase-8 into its active p18 and p10 protease subunits upon Fas-induced cell death. These results were confirmed by lower caspase-8 and caspase-3 protease activities in extracts of HeLa cells expressing HHV-8 v-FLIP. Coimmunoprecipitation studies further indicate that HHV-8 v-FLIP physically interacts with procaspase-8, but not with Fas-associated protein with death domain in the cellular cytoplasm. These results suggest that binding of HHV-8 v-FLIP to procaspase-8 affects the recruitment and the activation of the latter at the death-induced signaling complex, resulting in diminished apoptotic cascade initiation. Because cellular FLIP was recently reported to modulate promoter containing NF-kappaB motifs and that both HHV-8 and human immunodeficiency virus type 1 (HWV-1) can infect monocytes, we studied the effects of v-FLIP on HIV-1 gene expression. Cotransfection experiments indicated that v-FLIP expression is associated with activation of HIV long terminal repeats: events that were strictly dependent on the presence of NF-kappaB consensus elements. In conclusion, HHV-8 v-FLIP can possibly contribute to the pathogenesis of both HHV-8 and HIV-1 through impaired Fas-dependent killing of infected cells by cytotoxic T cells and through activation of HIV gene expression.

Amino Acid Sequence↗

PubMed2007

New way of regulating alternative splicing in retroviruses: the promoter makes a difference.

Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.

Alternative Splicing↗

PubMed1996

Protein kinase C-zeta mediates NF-kappa B activation in human immunodeficiency virus-infected monocytes.

The molecular mechanisms regulating human immunodeficiency virus (HIV) persistence in a major cell reservoir such as the macrophage remain unknown. NF-kappa B is a transcription factor involved in the regulation of the HIV long terminal repeat and is selectively activated following HIV infection of human macrophages. Although little information as to what signal transduction pathways mediate NF-kappa B activation in monocytes-macrophages is available, our previous work indicated that classical protein kinase C (PKC) isoenzymes were not involved in the HIV-mediated NF-kappa B activation. In this study, we have focused on atypical PKC isoenzymes. PKC-zeta belongs to this family and is known to be an important step in NF-kappa B activation in other cell systems. Immunoblotting experiments with U937 cells demonstrate that PKC-zeta is present in these cells, and its expression can be downmodulated by antisense oligonucleotides (AO). The HIV-mediated NF-kappa B activation is selectively reduced by AO to PKC-zeta. In addition, cotransfection of a negative dominant molecule of PKC-zeta (PKC-zeta mut) with NF-kappa B-dependent reporter genes selectively inhibits the HIV- but not phorbol myristate acetate- or lipopolysaccharide-mediated activation of NF-kappa B. That PKC-zeta is specific in regulating NF-kappa B is concluded from the inability of PKC-zeta(mut) to interfere with the basal or phorbol myristate acetate-inducible CREB- or AP1-dependent transcriptional activity. Lastly, we demonstrate a selective inhibition of p24 production by HIV-infected human macrophages when treated with AO to PKC-zeta. Altogether, these results suggest that atypical PKC isoenzymes, including PKC-zeta, participate in the signal transduction pathways by which HIV infection results in the activation of NF-kappa B in human monocytic cells and macrophages.

Base Sequence↗

PubMed2003

Nuclear factor kappa B: a potential target for anti-HIV chemotherapy.

The Nuclear Factor Kappa B (NF-kappaB) is a lymphoid-specific transcription factor, which is sequestered in the cytoplasm by the protein IkappaB. NF-kappaB plays a major role in the regulation of HIV-1 gene expression. Upon activation, NF-kappaB is released from IkappaB, moves to the nucleus, and binds to its sites on the HIV long terminal repeat to start transcription of integrated HIV genome. The present review focuses on the NF-kappaB as a potential target for the development of chemotherapy against HIV-1. Beginning from the viral-binding to reverse transcription, integration, and gene expression, to the virion maturation, the life cycle of HIV presents drug-targets at all the stages. As a result, many drugs have been developed and have entered clinical trials. Some of the most important of these are reverse transcriptase and protease inhibitors, which have been used mostly in clinical studies in the form of combined therapy. But, this combined therapy has presented the problem of resistance, due to mutations in the virus. However, targeting NF-kappaB for the suppression of virus does not present the problem of resistance, as NF-kappaB is a normal part of the human T-4 cell, and is not subject to mutations, as is the virus. An overview of the NF-kappaB system and its role in HIV-1 is presented, followed by a critical review of its current and potential synthetic inhibitors. The drugs studied against NF-kappaB fall mainly into three categories: (1) Antioxidants, against oxidative stress conditions, which aid in NF-kappaB activation, (2) IkappaB phosphorylation and degradation inhibitors (the phosphorylation and degradation of IkappaB is necessary to make NF-kappaB free and move to the nucleus), and (3) NF-kappaB DNA binding inhibitors. The antioxidants include N-Acetyl-L-cysteine (NAC), alpha-Lipoic acid, glutathione monoester, pyrrolidine dithiocarbamate, and tepoxalin, of which NAC is the best studied. The IkappaB phosphorylation and degradation inhibitors, which have been studied in the context of HIV-1 include the salicylates (sodium salicylate, and acetylsalicylic acid (aspirin)). Finally, the NF-kappaB DNA binding inhibitors, which have received attention only recently, are reviewed. These include the most potential, aurine tricarboxylic acid (ATA), a chelating agent, which has been found to inhibit NF-kappaB DNA binding at a low concentration of 30 micro M. The probable mechanism of action of these drugs is discussed alongwith relevant suggestions and conclusions.

Anti-HIV Agents↗

PubMed1991

Modulation of HIV-enhancer activity by heterologous agents: a minireview.

The enhancer region of the human immunodeficiency virus (HIV) long terminal repeat (LTR) contains two 10-bp (5'-GGGACTTTCC) repeats (core enhancers) which constitute the binding sites for the ubiquitous inducible cellular transcription factor, NF-kappa B. The NF-kappa B motifs of the LTR play a central role in transcriptional activation of the LTR by several heterologous viral proteins and various external chemical and physical stimuli. Activation of the HIV enhancer by these agents may lead to the onset of HIV gene expression resulting in active viral replication. Viral genes and chemical agents, which interfere with the activity of the enhancers may be useful in inhibiting HIV gene expression, thereby suppressing HIV replication.

Base Sequence↗

PubMed1992

Urocanic acid isomers, immunosuppressive cytokines and the induction of human immunodeficiency virus.

The ability of cis-urocanic acid to mimic the effects of ultraviolet (UV) was tested in two systems that show cellular responses to direct UV irradiation. First, cultured mouse keratinocytes were treated with cis-urocanic acid and the cell culture supernatants were injected into mice to test for the ability to block spleen cell proliferation in the mixed lymphocyte reaction. Second, human fibroblasts carrying the chloramphenicol acetyltransferase gene under the control of the human immunodeficiency virus (HIV) long terminal repeat promoter were tested for the ability of cis-urocanic acid to induce gene expression. In these tests cis-urocanic acid did not induce a response comparable with UV irradiation alone, although at toxic concentrations cis-UCA did induce low expression from the viral promoter. These data suggest that, unlike UVB radiation, cis-urocanic acid does not activate keratinocytes to produce immunosuppressive cytokines nor does it induce expression of the HIV promoter.

Animals↗

PubMed2003

Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.

Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR) occurs through the phosphorylation of the carboxy-terminal domain of the RNA polymerase II. The kinase complex, pTEFb, composed of cyclin T1 (CycT1) and CDK9, mediates this process. The trans-activation response (TAR) RNA-binding protein 2 (TRBP2) increases HIV-1 LTR expression through TAR and protein kinase R (PKR) binding, but not through interactions with the Tat-CycT1-CDK9 complex. TRBP2 and the Tat-CycT1-CDK9 complex have overlapping binding sites on TAR RNA. TRBP2 and CycT1 increased Tat trans-activation in NIH 3T3 cells with additive effects. Upon transfection of HIV-1 pLAI, pNL4-3, pMAL, and pAD molecular clones, reverse transcriptase (RT) activity and p24 concentration were decreased 200- to 900-fold in NIH 3T3 cells compared with HeLa cells in both cells and supernatants. In murine cells, cotransfection of the HIV clones with CycT1 or TRBP2 increased modestly the expression of RT activity in cell extracts. The analysis of Gag expression in murine cells transfected with CycT1 compared with human cells showed a 20-fold decrease in expression and a strong processing defect. The expression of both CycT1 and TRBP2 had a more than additive activity on RT function in cell extracts and on viral particle production in supernatant of murine cells. These results suggest an activity of CycT1 and TRBP2 at different steps in HIV-1 expression and indicate the requirement for another posttranscriptional factor in murine cells for full HIV replication.

Animals↗

PubMed2003

Stimulation of Tat-independent transcriptional processivity from the HIV-1 LTR promoter by matrix attachment regions.

The chromatin environment and the sites of integration in the host genome are critical determinants of human immunodeficiency virus (HIV) transcription and replication. Depending on the chromosomal location of provirus integration within the genome, HIV-1 long terminal repeat (LTR)-mediated transcription may vary from 0- to 70-fold. Cis-elements such as topoisomerase II cleavage sites, Alu repeats and matrix attachment regions (MARs) are thought to be targets for retroviral integration. Here we show that a novel MAR sequence from the T-cell receptor beta locus (MARbeta) and the IgH MAR mediate transcriptional augmentation when placed upstream of the HIV-1 LTR promoter. The effect of transcriptional augmentation is seen in both transient and stable transfection, indicating its effect even upon integration in the genome. MAR-mediated transcriptional elevation is independent of Tat, and occurs synergistically in the presence of Tat. Further, we show that MAR-mediated transcriptional elevation is specific to the HIV-1 LTR and the Moloney murine leukemia virus LTR promoter. In a transient transfection assay using over-expressed IkappaB, the inhibitor of NF-kappaB, we show that MAR-induced processive transcription is NF-kappaB dependent, signifying the role of local enhancers within the LTR promoter. Furthermore, by RNase protection experiments using proximal and distal probes, we show that MAR-mediated transcriptional upregulation is more prominent at the distal rather than the proximal end, thus indicating the potential role of MARs in promoting elongation.

Animals↗

PubMed1993

Differential inhibition of the DNA binding of transcription factors NF kappa B and OTF-1 by nitrogen mustard and quinacrine mustard: transcriptional implications.

Nitrogen mustard (HN2) and quinacrine mustard (QM) both inhibited the binding of NF kappa B to the GC-rich consensus sequence in the HIV long terminal repeat (LTR), as assessed by gel-shift assays. QM also inhibited the binding of OTF-1 to the AT-rich octamer present in the H2B promoter whereas HN2 was inactive. Inhibition of the binding of transcription factors was due to the drug interaction with DNA, since it also occurred when transcription factors were added to DNA after removal of free drug. In Jurkat cells transfected with pI3CAT, where the chloramphenicol acetyltransferase (CAT) gene is under the control of the HIV LTR, both HN2 and QM inhibited CAT gene expression. However, in Jurkat cells transfected with plasmid -147, where the CAT gene is under the control of the H2B promoter, QM inhibited CAT expression but HN2 did not. These results were obtained at concentrations of HN2 or QM that inhibited total DNA and RNA synthesis to a similar extent. The present results suggest that the more selective pharmacological activity of HN2 (HN2 is an active antineoplastic agent whereas QM is inactive and very toxic) might be related to its preferential functional inhibition of GC-rich consensus sequence, possibly important in the regulation of genes involved in the malignant proliferation and behavior of some tumors.

Base Composition↗

PubMed1994

Activation of HIV type 1 long terminal repeat by ultraviolet light is serum and strain specific.

We have studied the UV responsiveness of xeroderma pigmentosum (XP) and HeLa cell lines transfected with a CAT reporter gene under the control of the HIV-1 LTR promoter. XP fibroblasts grown in 10% newborn bovine serum (NBS) were three times more responsive to UV radiation than cells grown in 10% fetal calf serum (FCS). Moreover, cocultivation of UV-irradiated XP cells with XP cells containing stable integrants of HIV-LTR CAT was found to be more than four times more effective in inducing the CAT activity when cells were maintained in 10% NBS than in 10% FCS. The level of induction was also dependent on the serum concentration. These data indicate that a serum component, possibly a cytokine(s), can enhance the UV response of both irradiated cells and unirradiated cells cocultivated with irradiated cells. The magnitude of UV responsiveness seemed also to be strain dependent. CAT activity for the HIV LTR promoter from the HTLV-IIIB (HIV-IIIB) strain was induced more than 30-fold by UV irradiation whereas activity from the LAV-1BRU strain was less than 2-fold. In contrast, both constructs were strongly induced by Tat expression. This indicates that there are differences in the induction mechanism for these two stimuli, even though UV radiation has been previously reported to induce a cellular Tat-like factor (Valerie K, et al., Nature [London] 1988;333:78-81).

Base Sequence↗

PubMed1987

Activation of the human immunodeficiency virus by herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) and some of its immediate-early genes stimulate expression of the human immunodeficiency virus (HIV) long terminal repeat (LTR) sequences and the replication of HIV itself. To demonstrate this, the HIV LTR was linked to the indicator gene chloramphenicol acetyltransferase (CAT) and transfected into Vero cells with or without the trans-activating gene (tat) of HIV. Infection of these cells with HSV-1 strain KOS or temperature-sensitive mutant tsB21 or tsE6 resulted in a large increase in CAT activity in the absence of tat and further augmentation in the presence of tat. This stimulation was seen at both their permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures, implying either that HSV-1 infection or immediate-early gene expression is all that is required. In cotransfection assays in Vero cells, cloned HSV-1 immediate-early genes ICP0 and ICP4 stimulated CAT activity in the presence of tat, while ICP27 had no effect. On the other hand, in SW480 cells, ICP4 and, to a lesser extent, ICP0 genes caused stimulation of CAT activity in the absence of tat. Deletion mutants within the HIV LTR showed that the target for HSV stimulation is distinct from the tat-responsive area and maps near the SP1 binding sites. In Hela cells, ICP0 or ICP4 stimulated the replication of a cotransfected clone of HIV, as shown by an increase in reverse transcriptase activity in the culture supernatant.

Acetyltransferases↗

PubMed1991

Inhibition of transcription factors belonging to the rel/NF-kappa B family by a transdominant negative mutant.

The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.

Animals↗

PubMed1995

Regulation of macrophage activation and human immunodeficiency virus production by invasive Salmonella strains.

Salmonellae possess the ability to adhere to and invade macrophages and in so doing trigger a number of intracellular events that are associated with cellular activation. As an initial approach to defining the mechanisms by which invasive salmonellae alter macrophage function, we have explored the impact of Salmonella infection on the production of human immunodeficiency virus (HIV) in U1 cells, a promonocytic cell line latently infected with the virus. Infection of U1 cells with a pathogenic strain of Salmonella enteritidis resulted in a marked induction of macrophage activation and HIV production. The stimulatory effect of salmonellae was mediated by signals other than lipopolysaccharide. Salmonella mutants with specific defects in invasion or intracellular survival were markedly less effective in the induction of HIV production. In contrast to S. enteritidis, strains of Yersinia enterocolitica, Legionella pneumophila, and Escherichia coli did not induce HIV production. However, all of these bacteria induced comparable levels of gene expression mediated by the HIV long terminal repeat. The results of this study are consistent with the notion that invasive salmonellae possess the ability to activate the macrophage by at least one mechanism that is not shared with several other species of gram-negative bacteria. Furthermore, the expression of this unique property is maximal with Salmonella strains that are not only invasive but also capable of prolonged survival within the macrophage. Our results indicate that the U1 cell line may be a very useful model system with which to examine the biochemical pathways by which internalized salmonellae modulate the activation state of the macrophage.

Escherichia coli↗

PubMed2005

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia.

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.

Base Sequence↗

PubMed1995

An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction.

Adeno-associated virus (AAV) vectors are potentially useful for gene therapy of a number of human diseases. However, the use of these vectors has been limited by the lack of stable vector-packaging cell lines. The difficulties in developing packaging cell lines relate to low levels of rep gene expression from the AAV-p5 promoter, and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines. We describe here two new techniques which allow these problems to be circumvented. First, we have demonstrated that expression of rep from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter results in a 10-fold improvement of packaging efficiency. Second, we have overcome the inefficiency of vector plasmid transfection by generating cell populations containing rescuable AAV recombinant genomes. These improvements yielded a net increase of 50-fold in the packaging efficiency of the AAVp5neo and AAVp5lacZ recombinant vectors. The AAVp5lacZ vector packaged with this method was administered systemically to recently weaned C57BL mice, and mediated efficient expression of the beta-galactosidase reporter gene in cells of the airway epithelium and spleen. This indicates the in vivo activity of these vector stocks, and their potential utility for gene therapy.

Animals↗

PubMed1994

Identification of a novel cell-type and context specific enhancer within the negative regulatory element of the human immunodeficiency virus type 1 long terminal repeat.

One segment of the negative regulatory region of the human immunodeficiency virus long terminal repeat (HIV-LTR), nucleotides -273 to -255, is homologous to positive response elements for interleukin-2 and glucocorticoids. We demonstrate that this sequence, referred to as purine-rich response element (PRRE), acts as a classic enhancer in the context of a heterologous promoter, but serves as a functional repressor in the intact provirus, binding (a) cellular factor(s). These data suggest that certain DNA response elements in the HIV LTR serve as negative regulators only in the context of the intact LTR, presumably through interactions with other transcriptional factors, a phenomenon that might be exploited to interfere with viral gene transcription.

Base Sequence↗